Arginine deiminase with reduced cross-reactivity toward adi - peg 20 antibodies for cancer treatment

ABSTRACT

The present invention relates generally to isolated to arginine deiminase (ADI) proteins that have reduced cross-reactivity with anti-ADI-PEG 20 antibodies as compared to ADI-PEG 20, but which can have functional characteristics comparable to or better than ADI-PEG 20, compositions comprising the ADI proteins, and related methods of treating arginine-dependent diseases or related diseases such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 61/790,833, filed Mar. 15, 2013, which ishereby incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is POLA_003_01US_ST25.txt. The text file is about114 KB, was created on Mar. 14, 2014, and is being submittedelectronically via EFS-Web.

BACKGROUND Technical Field

The present invention relates generally to arginine deiminase (ADI)proteins, including ADI proteins having reduced cross-reactivity withADI-PEG 20 antibodies. Such ADI proteins are useful for treatingarginine-dependent or related diseases such as cancer.

Description of the Related Art

Amino acid deprivation therapy can be an effective treatment of someforms of cancer. To date, there is one known clinical example relevantto this approach which utilizes asparaginase to lower circulating levelsof asparagine and inhibit protein synthesis. This treatment isparticularly effective for acute lymphoblastic leukemia (Avramis 2005,Viera Pinheiro 2004). Acute lymphoblastic leukemia cells require theamino acid asparagine for growth and proliferation. In contrast, mostnormal human cells are capable of synthesizing asparagine and areunaffected by asparagine depletion. Therefore, decreasing serumasparagine with asparaginase can selectively kill the cancer cellswithout harming the normal cells, tissues, and host. An E. coli derivedform of asparaginase has been approved for human use. However,asparaginase is found only in microbes; which makes it highlyimmunogenic in humans and also has a short serum half-life followinginjection (Avramis 2005). To make asparaginase a more effective drug,these drawbacks were minimized by formulating the E. coli derivedasparaginase with polyethylene glycol (PEG) to reduce the immunogenicityof this enzyme and the associated allergic reactions. In addition, PEGgreatly prolongs the circulating half-life of asparaginase, whichreduces both the frequency of treatment and the total cost of thetherapy. PEG formulated asparaginase is approved for use and is marketedunder the trade name Oncaspar® (Oncaspar® 2011, Avramis 2005, VieraPinheiro 2004, Fu 2007, Zeidan 2008).

Arginine is another non-essential amino acid for humans and mice (forreview see Rogers 1994). In humans, arginine can be synthesized fromcitrulline in two steps via the Krebs (urea) cycle enzymesargininosuccinate synthetase (ASS, L-citrulline:L-aspartate ligase[AMP-forming], EC 6.3.4.5) and argininosuccinate lyase (ASL,L-argininosuccinate arginine-lyase, EC 4.3.2.) (Haines 2011, Wu 2009,Morris 2006, Husson 2003, Tapiero 2002, Rogers 1994). ASS catalyzes theconversion of citrulline and aspartic acid to argininosuccinate, whichis then converted to arginine and fumaric acid by ASL. An argininedeficient diet in humans does not evoke hyperammonemia, orotic aciduria,nor alter the rate of whole body nitric oxide (NO) synthesis in adulthumans (Tapiero 2002, Castillo 1995, Rogers 1994, Carey 1987, Barbul1986, Snyderman 1959, Rose 1949). Although preterm infants appear torequire arginine (Wu 2004), arginine levels do not correlate with ageamong infants, children and young adults (Lücke 2007). In 1992, Takakuand Sugimura separately reported that human melanomas and hepatocellularcarcinoma (HCC) cell lines appear to require arginine for growth. Otherstudies showed that pegylated ADI was effective for the treatment ofmelanomas and hepatomas with few adverse effects.

ADI-PEG 20 treatment requires multiple doses over a period of time.After a number of treatments, anti-ADI-PEG 20 antibodies can developthat may limit its continued effectiveness. Therefore, there is a needin the art for ADI that has reduced cross-reactivity to anti-ADI-PEG20antibodies for use in treatment in order to improve and extend theefficacy of arginine depletion therapy. The present invention providesthis and other advantages for the treatment of cancers.

References: Avramis V I, Panosyan E H. 2005. Clin Pharmacokinet44:367-393; Barbul A. 1986. J Parenteral Enteral Nutr 10:227-238; CareyG P, et al. 1987. J Nutr 117:1734-1739; Castillo L, et al. 1995. Am JPhysiol 268 (Endocrinol Metab 31):E360-367; Fu C H, Sakamoto K M. 2007.Expert Opin Pharmacother 8:1977-1984; Haines R J, et al. 2011. Int JBiochem Mol Biol 2:8-23; Husson A, et al. 2003. Eur J Biochem270:1887-1899; Lücke T, et al. 2007. Clin Chem Lab Med 45:1525-1530;Morris S M Jr. 2006. Am J Clin Nutr 83(Suppl):5985-5125; Rogers Q R.1994. In Proceedings from a Symposium Honoring Willard J. Visek—fromAmmonia to Cancer and Gene Expression. Special Publication 86—April,1994, Agriculture Experiment Station, University of Illinois, 211Mumford Hall, Urbana, Ill. 61801, pp. 9-21; Tapiero H, et al. 2002.Biomed Pharmacother 56:439-445, 2002; Viera Pinheiro J P, Boos J. 2004.Br J Haematol 125: 117-127; Wu G, et al. 2009. Amino Acids 37:153-168;Wu G, et al. 2004. J Nutr Biochem 15:442-451; Zeidan A, et al. 2008.Expert Opin Biol Ther 9:111-119).

BRIEF SUMMARY

One aspect of the present invention provides an isolated argininedeiminase, wherein the isolated arginine deiminase has reducedcross-reactivity with patient anti-ADI-PEG 20 antibodies. Also includedare therapeutic or pharmaceutical compositions comprising an isolatedarginine deiminase or a fragment thereof having ADI activity, and apharmaceutically-acceptable carrier. In certain embodiments, thecomposition is sterile and/or substantially free of pyrogens such asendotoxins. In one embodiment, the isolated arginine deiminase havingreduced cross-reactivity with patient anti-ADI-PEG 20 antibodies is notfrom M. hominis. In another embodiment, the isolated arginine deiminasehaving reduced cross-reactivity with patient anti-ADI-PEG 20 antibodiesis from an organism listed in Table 1. In certain embodiments theisolated arginine deiminase having reduced cross-reactivity with patientanti-ADI-PEG 20 antibodies has one or more properties comparable to orbetter than those of ADI-PEG 20. In this regard, the one or moreproperties includes, but is not limited to, Kcat, Km, pH optimum,stability, in vivo proteolytic stability, or no requirement for ions orcofactors that are not already present in blood, or any combinationthereof. In one embodiment, the isolated arginine deiminase havingreduced cross-reactivity with patient anti-ADI-PEG 20 antibodies, has atleast 20 surface residue changes as compared to M. hominis argininedeiminase. In another embodiment, the isolated arginine deiminase havingreduced cross-reactivity with patient anti-ADI-PEG 20 antibodies hasbetween 20 and 135 surface residue changes, between 40 and 100 surfaceresidue changes, between 30 and 60 surface residue changes, between 80and 100 surface residues changes, or between 100 and 120 surfaceresidues changes, as compared to M. hominis arginine deiminase.

In another embodiment, the isolated arginine deiminase having reducedcross-reactivity with patient anti-ADI-PEG 20 antibodies is from M.arginini, M. arthritidis, M. phocicerebrale, M. gateae, M. phocidae, M.columbinum, M. iowae, M. crocodyli, M. alligatoris, H. orenii, or M.bovis. Illustrative arginine deiminase having reduced cross-reactivitywith patient anti-ADI-PEG 20 antibodies comprise the amino acid sequenceset forth in any one of SEQ ID NOs:2-32.

In another embodiment, the isolated arginine deiminase having reducedcross-reactivity with patient anti-ADI-PEG 20 antibodies has beenmodified to remove at least one pegylation site. In another embodimentof the arginine deiminase having reduced cross-reactivity with patientanti-ADI-PEG 20 antibodies, at least one lysine residue has beenmodified by an amino acid substitution. In this regard, in certainembodiments, at least 5 lysine residues, at least 10 lysine residues, orat least 20 lysine residues have been modified by an amino acidsubstitution.

In another embodiment, the arginine deiminase having reducedcross-reactivity with patient anti-ADI-PEG 20 antibodies is covalentlybonded via a linker to a PEG molecule. In this regard, the argininedeiminase having reduced cross-reactivity with patient anti-ADI-PEG 20antibodies may be covalently bonded to one or more PEG molecule, such asto about 1 to about 10 or about 2 to about 8 PEG molecules. The PEGmolecules may be straight chain or branch chain PEG molecules and mayhave a total weight average molecular weight of from about 1,000 toabout 40,000, or a total weight average molecular weight of from about10,000 to about 30,000. In those embodiments where the PEG is covalentlybonded to the ADIr of the present invention, via a linker, the linkermay comprise a succinyl group, an amide group, an imide group, acarbamate group, an ester group, an epoxy group, a carboxyl group, ahydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, ahistidine group, a methylene group, or any combinations thereof. In oneembodiment, the source of the succinyl group is succinimidyl succinate.

Another aspect of the present invention provides a polynucleotideencoding an isolated arginine deiminase described herein, vectorscomprising the polynucleotide, and isolated host cells comprising thevectors.

An additional aspect of the present invention provides a compositioncomprising the isolated arginine deiminase having reducedcross-reactivity with patient anti-ADI-PEG 20 antibodies as describedherein and a physiologically acceptable carrier. In certain embodiments,the compositions further comprise a chemotherapeutic agent. Exemplarythemotherapeutic agents include, but are not limited to, docetaxel,carboplatin, cyclophosphamide, gemcitabine, cisplatin, sorafenib,sunitinib, and everolimus.

Another aspect of the present invention provides a method of treating,ameliorating the symptoms of, or inhibiting the progression of a cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a composition comprising the isolated argininedeiminase having reduced cross-reactivity with patient anti-ADI-PEG 20antibodies as described herein and a physiologically acceptable carrier,thereby treating, ameliorating the symptoms of, or inhibiting theprogression of the cancer. In certain embodiments, the patient in needthereof has been determined to have anti-ADI-PEG 20 antibodies. Inanother embodiment, the cancer is selected from the group consisting ofhepatocellular carcinoma, melanoma including metastatic melanoma,pancreatic cancer, prostate cancer, small cell lung cancer,mesothelioma, lymphocytic leukemia, chronic myelogenous leukemia,lymphoma, hepatoma, sarcoma, leukemia, acute myeloid leukemia, relapsedacute myeloid leukemia, breast cancer, ovarian cancer, colorectalcancer, gastric cancer, glioma, glioblastoma multiforme, non-small celllung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer,esophageal cancer, brain cancer, head and neck cancers, cervical cancer,testicular cancer, and stomach cancer.

Another aspect of the invention provides a method of treating,ameliorating the symptoms of, or inhibiting the progression of a cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a composition comprising ADI-PEG 20, and after aperiod of time, administering to the patient a composition comprisingthe isolated arginine deiminase having reduced cross-reactivity withpatient anti-ADI-PEG 20 antibodies as described herein and aphysiologically acceptable carrier, thereby treating, ameliorating thesymptoms of, or inhibiting the progression of the cancer. In thisregard, the period of time may be determined, for example, by detectinga predetermined level of anti-ADI-PEG 20 antibodies in the patientand/or measuring or otherwise observing ADI activity in the patient,wherein the composition comprising the isolated arginine deiminasehaving reduced cross-reactivity with patient anti-ADI-PEG 20 antibodiesis administered following detection of the predetermined level of saidanti-ADI-PEG 20 antibodies and/or measurement or observation of apredetermined level of ADI activity in the patient.

Also included are isolated arginine deiminase proteins described hereinfor use in the preparation or manufacture of a medicament for treating,ameliorating the symptoms of, or inhibiting the progression of a cancer.

DETAILED DESCRIPTION

Embodiments of the present invention relate to selected ADI enzymes,which in some embodiments are engineered to have a small number ofsurface lysine residues, and conjugated with PEG through a stablelinker. The selected ADI enzymes are chosen from a large number of ADIenzymes, from different organisms, based on their beneficial properties.These properties include the ability of the enzyme to establish andmaintain low arginine concentrations in human blood through ADIconversion of arginine to citrulline and ammonia. In addition, theselected ADI molecules have reduced cross-reactivity toward anti-ADI-PEG20 antibodies as compared to ADI-PEG 20, such antibodies possiblyresulting from a patient's previous treatment with ADI-PEG 20.

In certain embodiments, the enzymes in this invention are pegylated toprovide protection against renal clearance and proteolysis, as well asreduced immunogenicity or antigenicity. To increase the effectiveness ofthe pegylation, modifications to the enzymes may be engineered to reducethe number of surface lysine residues and therefore limit the number ofavailable PEG attachment sites. This provides more complete and uniformpegylation at the remaining lysine attachment residues.

The PEG linker selected to attach methoxy-PEG to ADI is chosen toprovide a chemically stable linkage. It is expected this will increasethe molecule's bioactive lifetime. A chemically stable linker will alsoeliminate hydrolysis and reduce an immune response that might occur to ade-pegylated linker attached to the enzyme surface.

These cumulative specifications result in one or more molecules thateffectively remove arginine from a patient's blood and are notneutralized or cleared by anti-ADI-PEG 20 antibodies from previousarginine depletion therapy. The molecules are pegylated so as to delayneutralization and clearance due to their own immunogenicity. Thesefactors will permit their use instead of ADI-PEG 20 or in addition toADI-PEG 20 (e.g., as a follow-on drug) to extend arginine depletiontherapy and therefore increase effectiveness of arginine depletiontreatment as an anti-cancer therapeutic.

Normal cells do not require arginine for growth, since they cansynthesize arginine from citrulline in a two step process catalyzed byASS and ASL. In contrast, certain cancers do not express ASS. Certaincancers do not express ASL, and other cancers may have diminishedexpression of, or may not express ASS and/or ASL. Therefore, thesecancers are auxotrophic for arginine. This metabolic difference may becapitalized upon to develop a safe and effective therapy to treat theseforms of cancer. ADI catalyzes the conversion of arginine to citrullinevia the arginine dihydrolase pathway, and may thus be used to eliminatearginine.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Current Protocols in ProteinScience, Current Protocols in Molecular Biology or Current Protocols inImmunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al.,Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995;Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rdEdition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual(1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984) and other like references.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur, if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

“Patient” or “subject” refers to an animal, in certain embodiments amammal, and in a specific embodiment, a human.

“Biocompatible” refers to materials or compounds which are generally notinjurious to biological functions and which will not result in anydegree of unacceptable toxicity, including allergenic and diseasestates.

The term “reference sequence” refers generally to a nucleic acid codingsequence, or amino acid sequence, to which another sequence is beingcompared. All polypeptide and polynucleotide sequences described hereinare included as references sequences, including those described by nameand those described in the Tables and the Sequence Listing.

Throughout the present disclosure, the following abbreviations may beused: PEG, polyethylene glycol; ADI, arginine deiminase; SS,succinimidyl succinate; SSA, succinimidyl succinimide; SPA, succinimidylpropionate; NHS, N-hydroxy-succinimide; ASS1 or ASS, argininosuccinatesynthetase; ASL, argininosuccinate lyase.

In the present invention, a polynucleotide encoding ADI may be derived,cloned, isolated, synthesized or produced from any source, including,for example, microorganisms, recombinant biotechnology or anycombination thereof. For example, arginine deiminase may be cloned frommicroorganisms of the genera Mycoplasma, Clostridium, Bacillus,Borrelia, Enterococcus, Streptococcus, Lactobacillus, and/or Giardia. Incertain embodiments, arginine deiminase is cloned from Mycoplasmaarthritidis, Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasmaarginini, Steptococcus pyogenes, Steptococcus pneumoniae, Borreliaburgdorferi, Borrelia afzelii, Giardia intestinalis, Clostridiumperfringens, Bacillus licheniformis, Enterococcus faecalis,Lactobacillus sake, or any combination thereof. In other embodiments,the arginine deiminase is cloned from a species listed in Table 1. Inparticular, the ADI used in the present invention may comprise the aminoacid sequence of any one of SEQ ID NOs: 1-32, or a variant or fragmentor extension thereof having ADI activity (e.g., able to metabolizearginine into citrulline and ammonia). Some of the sequences provided inthe sequence listing do not represent full-length ADI protein sequences.Thus, in certain embodiments, additional amino acid residues can beadded to either end of the sequences provided herein to make afull-length protein having ADI activity. The specific amino acids to beadded can be determined by the skilled person based on alignments ofknown ADI sequences. Such ADI molecules can be synthesized using knowntechniques. Illustrative “extended” ADI(r) are provided, for example, inSEQ ID NOs:26-32.

In certain embodiments, the ADI enzymes as described herein are comparedto the benchmark ADI-PEG 20 molecule derived from M. hominis. As usedherein, “ADI-PEG 20” refers to the ADI molecule known in the art anddescribed for example in U.S. Pat. Nos. 6,183,738; 6,635,462; Ascierto PA, et al. (2005) Pegylated arginine deiminase treatment of patients withmetastatic melanoma: results from phase I and II studies. J Clin Oncol23(30): 7660-7668; Izzo F, et al. (2004) Pegylated arginine deiminasetreatment of patients with unresectable hepatocellular carcinoma:results from phase I/II studies. J Clin Oncol 22(10): 1815-1822;Holtsberg F W, et al. (2002), Poly(ethylene glycol) (PEG) conjugatedarginine deiminase: effects of PEG formulations on its pharmacologicalproperties. J Control Release 80(1-3): 259-271; Kelly et al., (2012)British Journal of Cancer 106, 324-332. As would be recognized by theskilled artisan, this molecule is a pegylated (PEG 20,000) ADI enzymederived from M. hominis, and has two substitutions (K112E; P210S)relative to the wild type M. hominis ADI enzyme.

The arginine deiminase enzymes as described herein were screened from alarge number of ADI enzymes and have a reduced level of reactivity withanti-ADI-PEG 20 antibodies from patients. Anti-ADI-PEG 20 antibodies canappear in subjects treated with ADI-PEG 20 and can be measured usingknown methodologies. Reactivity to anti-ADI-PEG 20 antibodies can bedetermined for example using ELISA or other similar assays known to theskilled artisan.

In this regard, ADI-PEG 20 can be used as a comparison to assesscross-reactivity level to patient anti-ADI-PEG 20 antibodies. Across-reactivity level that is statistically significantly lower thanthat of ADI-PEG 20 to patient anti-ADI-PEG 20 antibodies may be usefulherein. In certain embodiments, the arginine deiminase enzymes asdescribed herein have low or no cross-reactivity to anti-ADI-PEG 20antibodies. In another embodiment, any reduction in reactivity toanti-ADI-PEG 20 antibodies as compared to reactivity with ADI-PEG 20 canbe beneficial as such an ADI enzyme would improve treatment options forpatients in need of arginine depletion therapy. Thus, the argininedeiminase enzymes as described herein have reduced cross-reactivity topatient anti-ADI-PEG 20 antibodies as compared to ADI-PEG 20 reactivityto such antibodies.

“ADIr” is used herein to refer to an ADI enzyme of the present inventionhaving reduced cross-reactivity to anti-ADI-PEG 20 antibodies ascompared to ADI-PEG 20 reactivity to such antibodies. “ADIr”nomenclature is used to distinguish the molecules identified herein fromADI and ADI-PEG 20 as known in the art.

The ADIr enzymes of the invention have characteristics or propertiescomparable to or better than those of ADI-PEG 20, in order to reduce andmaintain low blood arginine levels for effective cancer treatment. Suchproperties include Kcat, Km, pH optimum, stability, in vivo proteolyticstability and lack of requirement for ions or cofactors not alreadypresent in the blood, or any combination thereof. In certainembodiments, an ADIr as described herein has properties that are aboutor at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or higher, than comparable properties ofADI-PEG 20. In other embodiments an ADIr described herein has propertiesthat are about or at least about 100%, 105%, 110%, 120%, 140%, 150%,160%, 180%, 200%, 220%, 240%, 250%, 260%, 280%, 300%, 320%, 340, 350%,360%, 400%, 420%, 450%, 460%, 500%, 520%, 550% or higher than thespecific property of ADI-PEG 20 being compared.

Thus, in certain embodiments, an ADIr has a Kcat that is about or atleast about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% of the Kcat of ADI-PEG 20, or better. Incertain embodiments, an ADIr has a Kcat that is about or at least about100%, 105%, 110%, 120%, 125%, 140%, 150%, 160%, 180%, 200%, 220%, 240%,250%, 260%, 280%, 300%, 320%, 340, 350%, 360%, 400%, 420%, 450%, 460%,500%, 520%, 550% or higher, times that of the ADI-PEG 20 Kcat. Incertain embodiments, the Kcat of the ADIr enzymes described herein, orcompositions comprising same, is about 0.5 sec⁻¹ to about 15 sec⁻¹, andin a further embodiment, is from about 1 sec⁻¹ to about 12 sec⁻¹, about1 sec⁻¹ to about 10 sec⁻¹, about 1.5 sec⁻¹ to about 9 sec⁻¹, about 2sec⁻¹ to about 8 sec⁻¹ or about 2.5 sec⁻¹ to about 7 sec⁻¹. In certainembodiments, the ADIr or ADIr-PEG in a composition has a Kcat of about2.5 sec⁻¹ to about 7.5 sec⁻¹. In some embodiments, the ADIr or ADIr-PEGin a composition has a Kcat of about 2.5 sec⁻¹, about 3 sec⁻¹, about 3.5sec⁻¹, about 4 sec⁻¹, about 4.5 sec⁻¹, about 5 sec⁻¹, about 5.5 sec⁻¹,about 6 sec⁻¹, about 6.5 sec⁻¹, about 7 sec⁻¹, about 7.2 sec⁻¹, about7.5 sec⁻¹, about 8 sec⁻¹, about 10 sec⁻¹, about 15 sec⁻¹, about 20sec⁻¹, about 25 sec⁻¹, about 30 sec⁻¹, about 35 sec⁻¹, about 40 sec⁻¹,about 45 sec⁻¹, about 50 sec⁻¹, about 55 sec⁻¹, about 60 sec⁻¹, about 65sec⁻¹, about 70 sec⁻¹, about 75 sec⁻¹, about 80 sec⁻¹, about 85 sec⁻¹,about 90 sec⁻¹, about 95 sec⁻¹, or about 100 sec⁻¹.

Thus, in certain embodiments, an ADIr has a Km that is about or at leastabout 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the Km ofADI-PEG 20, or better. Thus, in certain embodiments, an ADIr has a Kmthat is about or at least about 100%, 105%, 110%, 120%, 130%, 140%,150%, 160%, 180%, 200%, 220%, 240%, or 250% that of the Km of ADI-PEG20. In one embodiment, an ADIr, or a pegylated formulation thereof, hasa Km of from about 0.5 μM to about 50 μM, or about 1.6 μM to about 48μM, or about 0.5 μM to about 15 μM, and in a further embodiment, is fromabout 1 μM to about 12 μM, about 1 μM to about 10 μM, about 1.5 μM toabout 9 μM, about 1.5 μM to about 8 μM or about 1.5 μM to about 7 μM. Incertain embodiments, the ADIr or ADIr-PEG in a composition has a Km ofabout 1.5 μM to about 6.5 μM. In some embodiments, the ADIr or pegylatedformulation thereof has a Km of about 1.5 μM, about 1.6 μM, about 2 μM,about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 8 μM,about 9 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16μM, about 18 μM about 20 μM, about 22 μM, about 24 μM, about 25 μM,about 26 μM, about 28 μM, about 30 μM, about 32 μM, about 34 μM, about35 μM, about 36 μM, about 38 μM, about 40 μM, about 42 μM, about 44 μM,about 45 μM, about 46 μM, about 48 μM, or about 50 μM.

In certain embodiments, an ADIr functions at a pH close to thephysiological pH of human blood. Thus, in one embodiment, an ADIrfunctions at a pH of about 4 to about 10.8, or about 6 to about 8, orabout 6.5 to about 7.5. In one embodiment, an ADIr has good enzymeactivity at about pH 7.4.

In certain embodiments, an ADIr has stability during long term storageand temperature and proteolytic stability during treatment in the humanbody. In further embodiments, an ADIr does not require ions or cofactorsfor activity that are not already present in blood.

In certain embodiments, an ADIr described herein generally has an aminoacid sequence sufficiently different from M. hominis so that there aresurface residue changes which will reduce or eliminate antigenic sitesfor anti-ADI-PEG 20 antibodies. In one embodiment, there will be nocross reactivity between the selected ADIr molecule and existinganti-ADI-PEG 20 antibodies in a subject, and a completely new immuneresponse will be generated in a subject rather than a maturation of theexisting response to M. hominis ADI. Thus, in one embodiment, an ADIr asdescribed herein has from 20%-85% sequence identity to M. hominis ADI asset forth in SEQ ID NO:1. In certain embodiments, an ADIr as describedherein has even lower percent sequence identity to M. hominis ADI, suchas 10% or 15% identity. In another embodiment, an ADIr as describedherein has 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% or even 83% identity to M.hominis ADI, and still has reduced cross-reactivity toward anti-ADI-PEG20 antibodies.

In one embodiment, an ADIr as described herein has from about 25-140surface residue changes as compared to M. hominis ADI. Surface residuescan be identified from the crystal structure of M. hominis ADI andsurface residues for ADI from other organisms can be determined bysequence homology. An ADIr as described herein may have about 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, orabout 140 surface residue changes as compared to M. hominis ADI (see SEQID NO:1).

In another embodiment, an ADIr as described herein has from about 25-140residue changes as compared to M. hominis ADI. Such residue changes neednot only be of surface amino acid residues. Such residue changes (oradditions or deletions) can be at either end of the molecule or may beat any residue of the ADI, such that the modified ADI has the desiredADI activity as described herein. Residues to be changed can beidentified from the crystal structure of M. hominis ADI and residues forADI from other organisms can be determined by sequence homology. An ADIras described herein may have about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, or about 140 amino acidresidue changes as compared to M. hominis ADI (see SEQ ID NO:1).

From a large number of ADI enzymes, Table 1 lists 24 ADIr enzymes withtheir sequence percent identity relative to M. hominis ADI. From theliterature, M. hominis, M. arginini, and M. arthritidis ADI amino acidsequences are closely related and these enzymes have good catalyticproperties. More recently, additional ADI enzymes have been discoveredthat have sequences closely related to these three. More distantlyrelated Mycoplasma ADI enzymes have been identified, although less isknown about them. And even more distantly related ADI enzymes frombacterial and other sources exist.

In certain embodiments, the ADIr enzymes identified herein from a numberof selected species, have surface lysine residues (in certainembodiments, up to 30 or more). However, in certain embodiments an ADIrenzyme may have many fewer surface lysine residues, such as just 2lysine residues as in the case of Mycobacterium bovis ADI, or even nolysine residues (see e.g., ADI from Mycobacterium sp. MCS; GenBank No.ABG10381). Therefore, the ADIr enzymes identified herein that havereduced cross-reactivity with anti-ADI-PEG 20 antibodies, have about 0,1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or more surfacelysine residues.

The terms “polypeptide,” “protein” and “peptide” are usedinterchangeably and mean a polymer of amino acids not limited to anyparticular length. The terms do not exclude modifications such asmyristoylation, sulfation, glycosylation, phosphorylation and additionor deletion of signal sequences. The terms “polypeptide” or “protein”means one or more chains of amino acids, wherein each chain comprisesamino acids covalently linked by peptide bonds, and wherein saidpolypeptide or protein can comprise a plurality of chains non-covalentlyand/or covalently linked together by peptide bonds, having the sequenceof native proteins, that is, proteins produced by naturally-occurringand specifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.The terms “polypeptide” and “protein” specifically encompass the ADIrproteins of the present disclosure, or sequences that have deletionsfrom, additions to, and/or substitutions of one or more amino acid ofthe ADIr proteins. In certain embodiments, the polypeptide is a“recombinant” polypeptide, produced by recombinant cell that comprisesone or more recombinant DNA molecules, which are typically made of ofheterologous polynucleotide sequences or combinations of polynucleotidesequences that would not otherwise be found in the cell.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldtypically be found in nature, (2) is essentially free of other proteinsfrom the same source, e.g., from the same species, (3) is expressed by acell from a different species, (4) has been separated from at leastabout 50 percent of polynucleotides, lipids, carbohydrates, or othermaterials with which it is associated in nature, (5) is not associated(by covalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein can be encoded by genomic DNA, cDNA,mRNA or other RNA, of may be of synthetic origin, or any combinationthereof. In certain embodiments, the isolated protein is substantiallyfree from proteins or polypeptides or other contaminants that are foundin its natural environment that would interfere with its use(therapeutic, diagnostic, prophylactic, research or otherwise).

The term “variant” includes a polypeptide that differs from a referencepolypeptide specifically disclosed herein (e.g., SEQ ID NOS:1-32) by oneor more substitutions, deletions, additions and/or insertions. Variantpolypeptides are biologically active, that is, they continue to possessthe enzymatic or binding activity of a reference polypeptide. Suchvariants may result from, for example, genetic polymorphism and/or fromhuman manipulation.

In many instances, a biologically active variant will contain one ormore conservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged. As described above,modifications may be made in the structure of the polynucleotides andpolypeptides of the present invention and still obtain a functionalmolecule that encodes a variant or derivative polypeptide with desirablecharacteristics.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their utility.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte & Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5). It is known inthe art that certain amino acids may be substituted by other amino acidshaving a similar hydropathic index or score and still result in aprotein with similar biological activity, i.e., still obtain abiological functionally equivalent protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 (specifically incorporated herein by reference in itsentirety), states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

A variant may also, or alternatively, contain non-conservative changes.In a preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of fewer than about 10,9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants mayalso (or alternatively) be modified by, for example, the deletion oraddition of amino acids that have minimal influence on theimmunogenicity, secondary structure, enzymatic activity, and/orhydropathic nature of the polypeptide.

In general, variants will display about or at least about 30%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% similarity or sequence identity or sequence homology to areference polypeptide sequence (e.g., SEQ ID NOS:1-32). Moreover,sequences differing from the native or parent sequences by the addition(e.g., C-terminal addition, N-terminal addition, both), deletion,truncation, insertion, or substitution of about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80,90, 100 or more amino acids but which retain the properties oractivities of a parent or reference polypeptide sequence arecontemplated.

In some embodiments, variant polypeptides differ from reference sequenceby at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3or 2 amino acid residue(s). In other embodiments, variant polypeptidesdiffer from a reference sequence by about or at least 0.5% or 1% butless than 20%, 15%, 10% or 5% of the residues. (If this comparisonrequires alignment, the sequences should be aligned for maximumsimilarity. “Looped” out sequences from deletions or insertions, ormismatches, are considered differences.).

The term “polypeptide fragment” refers to a polypeptide that has anamino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion or substitution of a naturally-occurring orrecombinantly-produced polypeptide. In certain embodiments, apolypeptide fragment can comprise an amino acid chain at least 5 toabout 400 amino acids long. It will be appreciated that in certainembodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350,or 400 amino acids long. Particularly useful polypeptide fragmentsinclude functional domains, including the catalytic ADI domains of theADIr described herein. In the case of an ADIr, useful fragments include,but are not limited to, the catalytic domain and the α-helical domain.

Many activated PEGs used for conjugation to ADI covalently bond tolysine residues. There are usually many fewer PEG molecules attached toADI than there are lysine residues. Both the number and distribution ofattachments can be heterogeneous from molecule to molecule. Anyparticular lysine residue will be modified in only a small fraction ofthe ADI molecules. This site modification heterogeneity and low PEGoccupancy can result in problems with both drug characterization and theeffectiveness of PEG shielding at antigenic sites. Therefore, in certainembodiments, the selected ADIr enzymes as described herein, are modifiedby lysine replacement with other residue types to reduce the number oflysine residues. This produces a more uniformly pegylated protein andincreases the PEG occupancy at the remaining lysine residues. Specificlysine residues chosen to be changed to other residues will be selectedin order to preserve enzyme activity. This more uniform pegylation isexpected to provide increased protection against proteolysis in bloodand increased shielding of antigenic sites from patient antibodies.

In certain embodiments, the ADIr of the present disclosure may bemodified as described in U.S. Pat. No. 6,635,462. In particular,modifications of one or more of the naturally occurring amino acidresidues of an ADIr can provide for an enzyme that is more easilyrenatured and formulated thereby improving the manufacture of ADIr andtherapeutic compositions comprising the same. In one embodiment, theADIr of the present disclosure is modified to remove one or more lysineresidues (e.g., the lysine can be substituted with another amino acid oranalogues thereof, or a non-natural amino acid). In particular, in oneembodiment, the ADIr is modified to be free of the lysine at a positionequivalent to 112, 374, 405 or 408 of SEQ ID NO:1 (M. hominis ADI), or acombination of one or more of these positions. In a further embodiment,the ADIr is modified to be free of one or more lysines, for example, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or more lysine residues, should they be present, can be substituted withanother amino acid or analogues thereof, or a nonnatural amino acid. Inone embodiment, an ADIr has 5 lysines substituted, for example, at anequivalent position to position 7, 88, 137, 209, and 380 of SEQ IDNO: 1. In another embodiment, an ADIr has 10 lysines substituted, forexample, at positions equivalent to positions 7, 9, 59, 88, 115, 116,137, 178, 209, and 380 of SEQ ID NO: 1. In yet another embodiment, anADIr has 15 lysines substituted, for example, at positions equivalent topositions 7, 9, 59, 66, 88, 91, 93, 115, 116, 137, 141, 178, 209, 279,and at position 380 of SEQ ID NO: 1. In one embodiment, an ADIrcomprises 21 lysines substituted, for example, at positions equivalentto positions 7, 9, 56, 59, 66, 88, 91, 93, 96, 115, 116, 137, 141, 178,209, 254, 279, 325, 326, 380, and 406 of SEQ ID NO: 1.

A native ADIr may be found in microorganisms and is immunogenic andrapidly cleared from circulation in a patient. These problems may beovercome by modifying an ADIr. Thus, the present disclosure providesADIr modified by a modifying agent, including, but not limited tomacromolecule polymers, proteins, peptides, polysaccharides, or othercompounds. Arginine deiminase as described herein and the modifyingagent may be linked by either covalent bonds or non-covalent interactionto form a stable conjugate or a stable composition to achieve a desiredeffect. In certain embodiments, the modified ADIr retains the biologicalactivity of an unmodified ADIr and has a longer half life in vivo andlower antigenicity than the unmodified, ADIr. In certain embodiments,the modified ADIr retains at least 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of thebiological activity of unmodified ADIr. Generally, the modified ADIrretains biological activity sufficient for therapeutic use.

In one embodiment, a modifying agent can be a polymer or a protein or afragment thereof that is biocompatible and can increase the half life ofADIr in blood. The modifying agent can be either chemically coupled toADIr or where applicable, linked to the ADIr via fusion proteinexpression.

Macromolecule polymers may include a non-peptide macromolecule polymer,which in certain embodiments, may have its own bioactivity. Suitablepolymers include, but are not limited to, polyenol compounds, polyethercompounds, polyvinylpyrrolidone, poly amino acids, copolymer of divinylether and maleic anhydride, N-(2-hydroxypropyl)-methacrylamide,polysaccharide, polyoxyethylated polyol, heparin or its fragment,poly-alkyl-ethylene glycol and its derivatives, copolymers ofpoly-alkyl-ethylene glycol and its derivatives, poly(vinyl ethyl ether),a,P-Poly[(2-hydroxyethyl)-DL-aspartamide], polycarboxylates, polyoxyethylene-oxymethylenes, polyacryloyl morpholines, copolymer of aminocompounds and oxyolefin, poly hyaluronic acid, polyoxiranes, copolymerof ethanedioic acid and malonic acid, poly (1,3-dioxolane), ethylene andmaleic hydrazide copolymer, poly sialic acid, cyclodextrin, etc. Incertain embodiments, the polymer is polyethylene glycol.

The polyenol compounds as used herein include, but are not limited to,polyethylene glycol (including monomethoxy polyethylene glycol,monohydroxyl polyethylene glycol), polyvinyl alcohol, polyallyl alcohol,polybutenol and the like, and their derivatives, such as lipids.

The polyether compounds include, but are not limited to poly alkyleneglycol (HO((CH2)_(x)O)_(n)H), polypropylene glycol, polyoxyrehylene(HO((CH₂)₂O)_(n)H), polyvinyl alcohol ((CH₂CHOH)_(n)).

Poly amino acids include, but are not limited to, polymers of one typeof amino acid or copolymers of two or more types of amino acids, forexample, polyalanine or polylysine, or block co-polymers thereof.

Polysaccharides include but are not limited to, glucosan and itsderivatives, for example dextran sulfate, cellulose and its derivatives(including methyl cellulose and carboxymethyl cellulose), starch and itsderivatives, polysucrose, etc.

In one specific embodiment of the present invention, ADIr is modified bycoupling with proteins or peptides, wherein one or more proteins orpeptides are directly or indirectly linked to ADIr. The proteins caneither be naturally existing proteins or their fragments, including butnot limited to naturally existing human serum proteins or theirfragments, such as thyroxine-binding protein, transthyretin, a1-acidglycoprotein, transferrin, fibrinogen, immunoglobulin, Ig Fc regions,albumin, and fragments thereof. By “fragment” is meant any portion of aprotein that is smaller than the whole protein but which retains thedesired function of the protein. The ADIr as described herein may bedirectly or indirectly linked to a protein via a covalent bond. Directlinking means that one amino acid of ADIr is directly linked to oneamino acid of the modifying protein, via a peptide bond or a disulfidebridge. Indirect linking refers to the linkages between a ADIr and amodifying protein, via originally existing chemical groups therebetweenor specific chemical groups added through biological or chemical means,or the combination of the above-mentioned linkages.

In one particular embodiment, ADIr is modified by covalent attachmentwith PEG. ADIr covalently modified with PEG (with or without a linker)may be hereinafter referred to as “ADIr-PEG.” When compared tounmodified ADIr, ADIr-PEG retains most of its enzymatic activity, is farless immunogenic or antigenic, has a greatly extended circulatinghalf-life, and is much more efficacious in the treatment of tumors.

“Polyethylene glycol” or “PEG” refers to mixtures of condensationpolymers of ethylene oxide and water, in a branched or straight chain,represented by the general formula H(OCH₂CH₂)nOH, wherein n is at least4. “Polyethylene glycol” or “PEG” is used in combination with a numericsuffix to indicate the approximate weight average molecular weightthereof. For example, PEG5,000 refers to PEG having a total weightaverage molecular weight of about 5,000; PEG12,000 refers to PEG havinga total weight average molecular weight of about 12,000; and PEG20,000refers to PEG having a total weight average molecular weight of about20,000.

In one embodiment of the present invention, the PEG has a total weightaverage molecular weight of about 1,000 to about 50,000; in oneembodiment from about 3,000 to about 40,000, and in another embodimentfrom about 5,000 to about 30,000; in certain embodiments from about8,000 to about 30,000; in other embodiments from about 11,000 to about30,000; in additional embodiments, from about 12,000 to about 28,000; instill other embodiments, from about 16,000 to about 24,000; and in otherembodiments, about 18,000 to about 22,000; in another embodiment, from19,000 to about 21,000, and in one embodiment, the PEG has a totalweight average molecular weight of about 20,000. Generally, PEG with amolecular weight of 30,000 or more is difficult to dissolve, and yieldsof the formulated product may be reduced. The PEG may be a branched orstraight chain. Generally, increasing the molecular weight of the PEGdecreases the immunogenicity of the ADIr. The PEG having a molecularweight described in this embodiment may be used in conjunction withADIr, and, optionally, a biocompatible linker, to treat cancer,including, for example, acute myeloid leukemia, such as relapsed acutemyeloid leukemia, breast cancer, ovarian cancer, colorectal cancer,gastric cancer, glioma, glioblastoma multiforme, non-small cell lungcancer (NSCLC), kidney cancer, bladder cancer, uterine cancer,esophageal cancer, brain cancer, head and neck cancers, cervical cancer,testicular cancer, stomach cancer and esophageal cancer.

In another embodiment of the present invention, the PEG has a totalweight average molecular weight of about 1,000 to about 50,000; incertain embodiments about 3,000 to about 30,000; in other embodimentsfrom about 3,000 to about 20,000; in one embodiment from about 4,000 toabout 12,000; in still other embodiments from about 4,000 to about10,000; in additional embodiments from about 4,000 to about 8,000; stillfurther embodiments from about 4,000 to about 6,000; and about 5,000 inanother embodiment. The PEG may be a branched or straight chain, and incertain embodiments is a straight chain. The PEG having a molecularweight described in this embodiment may be used in conjunction withADIr, and optionally, a biocompatible linker, to treat graft versus hostdisease (GVHD) or cancer.

While ADIr-PEG is the illustrative modified ADIr described herein, aswould be recognized by the skilled person ADIr may be modified withother polymers or appropriate molecules for the desired effect, inparticular reducing antigenicity and increasing serum half-life.

ADIr may be covalently bonded to a modifying agent, such as PEG, with orwithout a linker, although a preferred embodiment utilizes a linker.

The linker used to covalently attach ADIr to a modifying agent, e.g.PEG, may be any biocompatible linker. As discussed above,“biocompatible” indicates that the compound or group is non-toxic andmay be utilized in vitro or in vivo without causing injury, sickness,disease, or death. A modifying agent, such as PEG, can be bonded to thelinker, for example, via an ether bond, a thiol bond, or an amide bond.The linker group includes, for example, a succinyl group, an amidegroup, an imide group, a carbamate group, an ester group, an epoxygroup, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosinegroup, a cysteine group, a histidine group, a methylene group, andcombinations thereof. In one embodiment, the source of the biocompatiblelinker is succinimidyl succinate (SS). Other suitable sources of linkermay include an oxycarbonylimidazole group (including, for example,carbonylimidazole (CDI), a nitro phenyl group (including, for example,nitrophenyl carbonate (NCP) or trichlorophenyl carbonate (TCP)), atrysylate group, an aldehyde group, an isocyanate group, a vinylsulfonegroup, or a primary amine. In another embodiment, the linker is derivedfrom SS, SPA, SCM, or NHS; in certain embodiments, SS, SPA, or NHS areused, and in other embodiments, SS or SPA are used. Thus, in certainembodiments, potential linkers can be formed from methoxy-PEGsuccinimidyl succinate (SS), methoxy-PEG succinimidyl glutarate (SG),methoxy-PEG succinimidyl carbonate (SC), methoxy-PEG succinimidylcarboxymethyl ester (SCM), methoxy-PEG2 N-hydroxy succinimide (NHS),methoxy-PEG succinimidyl butanoate (SBA), methoxy-PEG succinimidylpropionate (SPA), methoxy-PEG succinimidyl glutaramide, and methoxy-PEGsuccinimidyl succinamide.

Alternatively, ADIr may be coupled directly to a modifying agent, suchas PEG (i.e., without a linker) through an amino group, a sulfhydrylgroup, a hydroxyl group or a carboxyl group.

ADIr may be covalently bonded to PEG, via a biocompatible linker, usingmethods known in the art, as described, for example, by Park et al,Anticancer Res., 1:373-376 (1981); and Zaplipsky and Lee, PolyethyleneGlycol Chemistry: Biotechnical and Biomedical Applications, J. M.Harris, ed., Plenum Press, NY, Chapter 21 (1992), the disclosures ofwhich are hereby incorporated by reference herein in their entirety.

The attachment of PEG to ADIr increases the circulating half-life ofADIr. Generally, PEG is attached to a primary amine of ADIr. Selectionof the attachment site of PEG, or other modifying agent, on the ADIr isdetermined by the role of each of the sites within the active domain ofthe protein, as would be known to the skilled artisan. PEG may beattached to the primary amines of ADIr without substantial loss ofenzymatic activity. For example, the lysine residues present in ADIr areall possible points at which ADIr as described herein can be attached toPEG via a biocompatible linker, such as SS, SPA, SCM, SSA and/or NHS.PEG may also be attached to other sites on ADIr, as would be apparent toone skilled in the art in view of the present disclosure.

From 1 to about 30 PEG molecules may be covalently bonded to ADIr. Incertain embodiments, ADIr is modified with one PEG molecule. In otherembodiments, ADIr is modified with more than one PEG molecule. In oneembodiment, ADIr is modified with about 1 to about 10, or from about 7to about 15 PEG molecules, and in one embodiment from about 2 to about 8or about 9 to about 12 PEG molecules. In another embodiment, the ADIr ismodified with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEGmolecules. In one specific embodiment, ADIr is modified with 4.5-5.5 PEGmolecules per ADIr. In another embodiment, ADIr is modified with 5±1.5PEG molecules.

In another embodiment, about 15% to about 70% of the primary aminogroups in ADIr are modified with PEG, in one embodiment about 20% toabout 65%, about 25% to about 60%, or in certain embodiments about 30%to about 55%, or 45% to about 50%, and in other embodiments about 50% ofthe primary amino groups in arginine deiminase are modified with PEG.When PEG is covalently bonded to the end terminus of ADIr, it may bedesirable to have only 1 PEG molecule utilized. Increasing the number ofPEG units on ADIr increases the circulating half life of the enzyme.However, increasing the number of PEG units on ADIr decreases thespecific activity of the enzyme. Thus, a balance needs to be achievedbetween the two, as would be apparent to one skilled in the art in viewof the present disclosure.

In the present invention, a common feature of biocompatible linkers isthat they attach to a primary amine of arginine deiminase via asuccinimide group. Once coupled with ADIr, SS-PEG has an ester linkagenext to the PEG, which may render this site sensitive to serum esterase,which may release PEG from ADIr in the body. SPA-PEG and PEG2-NHS do nothave an ester linkage, so they are not sensitive to serum esterase.

In certain embodiments, a biocompatible linker is used in the presentinvention. PEG which is attached to the protein may be either a straightchain, as with SS-PEG, SPA-PEG and SC-PEG, or a branched chain of PEGmay be used, as with PEG2-NHS.

In certain embodiments, pegylation sites associated with ADIr located ator adjacent to the catalytic region of the enzyme are modified. Forpurposes of the present invention, the phrase “pegylation site” may bedefined as any site or position of ADI or a ADIr that may be covalentlymodified with polyethylene glycol. A “pegylation site” can be consideredlocated at or adjacent the catalytic region of the enzyme wherepegylation of the site results in a significant reduction in catalyticactivity of the enzyme. The pegylation of such sites has traditionallyresulted in the inactivation of the enzyme. For example, ADI fromMycoplasma hominis has a lysine at the 112 position which can beconsidered to be at or adjacent the catalytic region of the enzyme. Theattachment of PEG to this lysine at the 112 position can inactivate theenzyme. In addition, ADI from Mycoplasma hominis has a cysteine at the397 position which can be considered to be at or adjacent the catalyticregion of the enzyme. The amino acid substitutions for cysteine at the397 position can inactivate the enzyme. In particular, substitutingalanine, histidine, arginine, serine, lysine or tyrosine for cysteine atthe 397 position can result in a loss of all detectable enzyme activity.ADI from Mycoplasma hominis also has three lysines located near thisconserved cysteine, in particular Lys374, Lys405 and Lys408. Theattachment of PEG to Lys374, Lys405, Lys408 or combinations thereof caninactivate the enzyme.

It is to be understood that ADIr derived from other organisms may alsohave pegylation sites corresponding to 112 position of ADI fromMycoplasma hominis. In addition, ADI from some organisms may havelysines corresponding to the same general location as the 112 positionof ADI from Mycoplasma hominis. The location of lysine in ADI from suchorganisms are known to the skilled person and are described in U.S. Pat.No. 6,635,462.

Thus, in one embodiment, the present invention provides for certainamino acid substitutions in the polypeptide chain of ADIr. These aminoacid substitutions provide for modified ADIr that loses less activitywhen modified by a modifying agent, e.g., upon pegylation. Byeliminating pegylation sites, or other known modification sites, at oradjacent to the catalytic region of enzyme, optimal modification, e.g.,pegylation, can be achieved without the loss of activity.

It is to be understood that other embodiments of the invention are basedon the understanding that certain structural characteristics of argininedeiminase may prevent or interfere with the proper and rapidrenaturation when produced via recombinant technology. In particular,these structural characteristics hinder or prevent the enzyme fromassuming an active conformation during recombinant production. Forpurposes of the present invention, the phrase “active conformation” maybe defined as a three-dimensional structure that allows for enzymaticactivity by unmodified or modified arginine deiminase. The activeconformation may, in particular, be necessary for catalyzing theconversion of arginine into citrulline. The phrase “structuralcharacteristic” may be defined as any trait, quality or property of thepolypeptide chain resulting from a particular amino acid or combinationof amino acids. For instance, arginine deiminase may contain an aminoacid that results in a bend or kink in the normal peptide chain and thushinders the enzyme from assuming an active conformation duringrenaturation of the enzyme. In particular, arginine deiminase fromMycoplasma hominis has a proline at the 210 position that may result ina bend or kink in the peptide chain, making it more difficult torenature the enzyme during recombinant production. It is to beunderstood that arginine deiminase derived from other organisms may alsohave sites corresponding to the 210 position of arginine deiminase fromMycoplasma hominis.

The present invention thus again provides for certain amino acidsubstitutions in the polypeptide chain of wild type arginine deiminases.Such amino acid substitutions can eliminate the problematic structuralcharacteristics in the peptide chain of arginine deiminase. Such aminoacid substitutions provide for improved renaturation of the modifiedarginine deiminase. These amino acid substitutions make possible rapidrenaturing of modified arginine deiminases using reduced amounts ofbuffer. These amino acid substitutions may also provide for increasedyields of renatured modified arginine deiminase. In one embodiment ofthe invention, the modified arginine deiminase has an amino acidsubstitution at P210 or the equivalent residue. As mentioned above,arginine deiminase derived from Mycoplasma hominis has the amino acidproline located at the 210 position. While not limiting the presentinvention, it is presently believed that the presence of the amino acidproline at position 210 results in a bend or kink in the normalpolypeptide chain that increases the difficulty of renaturing (i.e.,refolding) arginine deiminase. Substitutions for proline at position 210make possible the rapid renaturation of modified arginine deiminaseusing reduced amounts of buffer. Substitutions for proline at position210 may also provide for increased yields of renatured modified argininedeiminase. In one embodiment, the proline at position 210 is substitutedwith serine. It is to be understood that in accordance with this aspectof the invention, other substitutions at position 210 may be made.Examples of other substitutions include Pro210 to Thr210, Pro210 toArg210, Pro210 to Asn210, Pro210 to Gln210 or Pro210 to Met210. Byeliminating those structural characteristics associated with the aminoacid of position 210 of the wild-type arginine deiminase, properrefolding of the enzyme can be achieved.

The methods of the present invention can involve either in vitro or invivo applications. In the case of in vitro applications, including cellculture applications, the compounds described herein can be added to thecells in cultures and then incubated. The compounds of the presentinvention may also be used to facilitate the production of monoclonaland/or polyclonal antibodies, using antibody production techniques wellknown in the art. The monoclonal and/or polyclonal antibodies can thenbe used in a wide variety of diagnostic applications, as would beapparent to one skilled in the art.

The in vivo means of administration of the compounds of the presentinvention will vary depending upon the intended application.Administration of the ADIr compositions described herein, in pure formor in an appropriate pharmaceutical composition, can be carried out viaany of the accepted modes of administration of agents for servingsimilar utilities. The pharmaceutical compositions can be prepared bycombining ADIr, e.g., ADIr-PEG, ADIr-PEG 20, with an appropriatephysiologically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. In addition, other pharmaceutically active ingredients(including other anti-cancer agents as described elsewhere herein)and/or suitable excipients such as salts, buffers and stabilizers may,but need not, be present within the composition. Administration may beachieved by a variety of different routes, including oral, parenteral,nasal, intravenous, intradermal, subcutaneous or topical. Modes ofadministration depend upon the nature of the condition to be treated orprevented. Thus, ADIr-PEG, e.g., ADIr-PEG 20, may be administeredorally, intranasally, intraperitoneally, parenterally, intravenously,intralymphatically, intratumorly, intramuscularly, interstitially,intra-arterially, subcutaneously, intraocularly, intrasynovial,transepithelial, and transdermally. An amount that, followingadministration, reduces, inhibits, prevents or delays the progressionand/or metastasis of a cancer is considered effective. In certainembodiment, the ADIr compositions herein increase median survival timeof patients by a statistically significant amount. In one embodiment,the ADIr treatments described herein increase median survival time of apatient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. Incertain embodiments, ADIr treatments increase median survival time of apatient by 1 year, 2 years, 3 years, or longer. In one embodiment, theADIr treatments described herein increase progression-free survival by 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10weeks or longer. In certain embodiments, the ADIr treatments describedherein increase progression-free survival by 1 year, 2 years, 3 years,or longer.

In certain embodiments, the amount administered is sufficient to resultin tumor regression, as indicated by a statistically significantdecrease in the amount of viable tumor, for example, at least a 10%,20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered(e.g., decreased with statistical significance) scan dimensions. Incertain embodiments, the amount administered is sufficient to result instable disease. In other embodiments, the amount administered issufficient to result in clinically relevant reduction in symptoms of aparticular disease indication known to the skilled clinician.

In certain embodiments the amount administered is sufficient to inhibitNO synthesis, inhibit angiogenesis, and or is sufficient to induceapoptosis in tumor cells or any combination thereof. NO synthesis,angiogenesis and apoptosis may be measured using methods known in theart, see, e.g., Current Protocols in Immunology or Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y. (2009 and updatesthereto); Ausubel et al., Short Protocols in Molecular Biology, 3^(rd)ed., Wiley & Sons, 1995; and other like references. In one particularembodiment the amount administered inhibits NO synthesis and inhibitsthe growth of melanoma and complements, adds to, or synergizes withother chemotherapies as described herein, such as cisplatin.Accordingly, one embodiment of the present disclosure provides a methodof treating melanoma by administering ADIr-PEG 20 in combination withcisplatin, wherein the treatment depletes endogenous nitric oxide (NO).

The precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by testing the compositions in model systems knownin the art and extrapolating therefrom. Controlled clinical trials mayalso be performed. Dosages may also vary with the severity of thecondition to be alleviated. A pharmaceutical composition is generallyformulated and administered to exert a therapeutically useful effectwhile minimizing undesirable side effects. The composition may beadministered one time, or may be divided into a number of smaller dosesto be administered at intervals of time. For any particular subject,specific dosage regimens may be adjusted over time according to theindividual need.

The ADIr compositions may be administered alone or in combination withother known cancer treatments, such as radiation therapy, chemotherapy,transplantation, immunotherapy, hormone therapy, photodynamic therapy,etc. The compositions may also be administered in combination withantibiotics.

The ADIr compositions may also be administered alone or in combinationwith ADI-PEG 20 therapy. In certain embodiments, the ADIr as describedherein are used in patients who have been treated with ADI-PEG 20 andwho have developed anti-ADI-PEG 20 antibodies. Such patients no longerbenefit from ADI-PEG 20 treatment as the enzyme is neutralized by theantibodies. Thus, in certain embodiments, the invention provides amethod of treating, ameliorating the symptoms of, or inhibiting theprogression of a cancer comprising administering to a patient in needthereof a therapeutically effective amount of a composition comprisingADI-PEG 20, and after a period of time, administering to the patient acomposition comprising an ADIr as described herein, thereby treating,ameliorating the symptoms of, or inhibiting the progression of thecancer.

In one embodiment of the method, the period of time is determined bydetecting a predetermined level of anti-ADI-PEG 20 antibodies in thepatient, wherein the composition comprising an ADIr is administeredfollowing detection of the predetermined level of said anti-ADI-PEG 20antibodies. In certain embodiments, threshold level(s) or predeterminedlevels of anti-ADI-PEG 20 antibodies in patients to be treated withADI-PEG 20 and the ADIr of the present invention can be established. A“predetermined threshold level” (also referred to as “predeterminedlevel” or “predetermined cut-off value”), or sometimes referred to as apredetermined cut off, of anti-ADI-PEG 20 antibodies may be establishedusing methods known in the art, for example, using Receiver OperatorCharacteristic curves or “ROC” curves. In one embodiment, even very lowlevels of anti-ADI-PEG 20 antibodies is deemed sufficient to warrantswitching treatment from ADI-PEG 20 to an ADIr-PEG of the presentinvention. In certain embodiments, an appropriate level of anti-ADI-PEG20 that will determine when to terminate ADI-PEG 20 treatment and begintreatment with an ADIr-PEG of the present invention can be determined bythe skilled clinician.

In some embodiments, the period of time is determined by detecting orotherwise observing ADI activity in the patient, wherein the compositionis administered following detection or observation of a predeterminedlevel of ADI activity. In particular embodiments, the composition isadministered following detection or observation of a reduced level ofADI activity in the patient. ADI activity can be measured directly, forexample, by assaying a biological sample for at least one indicator ofADI activity, or indirectly, for example, by observing the desired orintended effect of the ADI-PEG 20 treatment. In certain embodiments, anappropriate level of ADI activity that will determine when to terminateADI-PEG 20 treatment and begin treatment with an ADIr-PEG of the presentinvention can be determined by the skilled clinician.

Typical routes of administering these and related pharmaceuticalcompositions thus include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques.

Pharmaceutical compositions according to certain embodiments of thepresent invention are formulated so as to allow the active ingredientscontained therein to be bioavailable upon administration of thecomposition to a patient. Compositions that will be administered to asubject or patient may take the form of one or more dosage units, wherefor example, a tablet may be a single dosage unit, and a container of aherein described ADIr composition in aerosol form may hold a pluralityof dosage units. Actual methods of preparing such dosage forms areknown, or will be apparent, to those skilled in this art; for example,see Remington: The Science and Practice of Pharmacy, 20th Edition(Philadelphia College of Pharmacy and Science, 2000). The composition tobe administered will, in any event, contain a therapeutically effectiveamount of an ADIr-PEG of the present disclosure, such as ADIr-PEG 20,for treatment of a disease or condition of interest in accordance withteachings herein. In certain embodiments, the pharmaceutical ortherapeutic compositions are sterile and/or pyrogen-free.

A pharmaceutical composition may be in the form of a solid or liquid. Inone embodiment, the carrier(s) are particulate, so that the compositionsare, for example, in tablet or powder form. The carrier(s) may beliquid, with the compositions being, for example, anoral oil, injectableliquid or an aerosol, which is useful in, for example, inhalatoryadministration. When intended for oral administration, thepharmaceutical composition is generally either solid or liquid form,where semi-solid, semi-liquid, suspension and gel forms are includedwithin the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like. Such a solid compositionwill typically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following may be present: binders such ascarboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gumtragacanth or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin; a flavoring agent such as peppermint,methyl salicylate or orange flavoring; and a coloring agent. When thepharmaceutical composition is in the form of a capsule, for example, agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of the followingadjuvants: sterile diluents such as water for injection, salinesolution, in certain embodiments, physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition intended for either parenteral ororal administration should contain an amount of ADIr as hereindisclosed, such as ADIr-PEG 20, such that a suitable dosage will beobtained. Typically, this amount is at least 0.01% of ADIr in thecomposition. When intended for oral administration, this amount may bevaried to be between 0.1 and about 70% of the weight of the composition.Certain oral pharmaceutical compositions contain between about 4% andabout 75% of ADIr-PEG. In certain embodiments, pharmaceuticalcompositions and preparations according to the present invention areprepared so that a parenteral dosage unit contains between 0.01 to 10%by weight of ADIr-PEG prior to dilution.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. The pharmaceutical composition may beintended for rectal administration, in the form, for example, of asuppository, which will melt in the rectum and release the drug. Thecomposition for rectal administration may contain an oleaginous base asa suitable nonirritating excipient. Such bases include, withoutlimitation, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition may include various materials, whichmodify the physical form of a solid or liquid dosage unit. For example,the composition may include materials that form a coating shell aroundthe active ingredients. The materials that form the coating shell aretypically inert, and may be selected from, for example, sugar, shellac,and other enteric coating agents. Alternatively, the active ingredientsmay be encased in a gelatin capsule. The pharmaceutical composition insolid or liquid form may include an agent that binds to ADIr-PEG andthereby assists in the delivery of the compound. Suitable agents thatmay act in this capacity include monoclonal or polyclonal antibodies,one or more proteins or a liposome. The pharmaceutical composition mayconsist essentially of dosage units that can be administered as anaerosol. The term aerosol is used to denote a variety of systems rangingfrom those of colloidal nature to systems consisting of pressurizedpackages. Delivery may be by a liquefied or compressed gas or by asuitable pump system that dispenses the active ingredients. Aerosols maybe delivered in single phase, bi-phasic, or tri-phasic systems in orderto deliver the active ingredient(s). Delivery of the aerosol includesthe necessary container, activators, valves, subcontainers, and thelike, which together may form a kit. One of ordinary skill in the art,without undue experimentation may determine preferred aerosols.

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art. For example, a pharmaceuticalcomposition intended to be administered by injection can be prepared bycombining a composition that comprises ADIr-PEG as described herein andoptionally, one or more of salts, buffers and/or stabilizers, withsterile, distilled water so as to form a solution. A surfactant may beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact withthe ADIr-PEG composition so as to facilitate dissolution or homogeneoussuspension of the ADIr-PEG in the aqueous delivery system.

The compositions may be administered in a therapeutically effectiveamount, which will vary depending upon a variety of factors includingthe activity of the specific compound (e.g., ADIr-PEG) employed; themetabolic stability and length of action of the compound; the age, bodyweight, general health, sex, and diet of the patient; the mode and timeof administration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy.

A therapeutically effective amount of one of the compounds of thepresent invention is an amount that is effective to inhibit tumorgrowth. Generally, treatment is initiated with small dosages which canbe increased by small increments until the optimum effect under thecircumstances is achieved. Generally, a therapeutic dosage of compoundsof the present invention may be from about 1 to about 200 mg/kg twice aweek to about once every two weeks. For example, the dosage may be about1 mg/kg once a week as a 2 ml intravenous injection to about 20 mg/kgonce every 3 days. In a further embodiment, the dose may be from about50 IU/m² to about 700 IU/m², administered about once every 3 days, aboutonce a week, about twice a week, or about once every 2 weeks. In certainembodiments, the dose may be about 50 IU/m², 60 IU/m², 70 IU/m², 80IU/m², 90 IU/m², 100 IU/m², 110 IU/m², 120 IU/m², 130 IU/m², 140 IU/m²150 IU/m², 160 IU/m², 170 IU/m², 180 IU/m², 190 IU/m², 200 IU/m², 210IU/m², 220 IU/m², 230 IU/m², 240 IU/m², 250 IU/m², 260 IU/m², 270 IU/m²,280 IU/m², 290 IU/m², 300 IU/m², 310 IU/m², about 320 IU/m², about 330IU/m², 340 IU/m² about 350 IU/m² 360 IU/m², 370 IU/m², 380 IU/m², 390IU/m², 400 IU/m², 410 IU/m², 420 IU/m², 430 IU/m², 440 IU/m², 450 IU/m²,500 IU/m², 550 IU/m², 600 IU/m², 620 IU/m², 630 IU/m², 640 IU/m², 650IU/m², 660 IU/m², 670 IU/m², 680 IU/m², 690 IU/m², or about 700 IU/m²administered about once every 3 days, about once a week, about twice aweek, or about once every 2 weeks. In certain embodiments, the dose maybe modified as desired by the skilled clinician.

The optimum dosage with ADIr-SS-PEG5,000 may be about twice a week,while the optimum dosage with ADIr-SS-PEG20,000 may be from about once aweek to about once every two weeks. In certain embodiments, the optimumdosage with ADIr-SS-PEG20,000 may be about twice a week.

ADIr-PEG may be mixed with a phosphate buffered saline solution, or anyother appropriate solution known to those skilled in the art, prior toinjection. In one embodiment, a liquid composition comprising ADIr-PEGcomprises about 10 to about 12 mg of ADIr, about 20 to about 40 mg ofpolyethylene glycol, 1.27 mg+5% monobasic sodium phosphate, USP; about 3mg+5% dibasic sodium phosphate, USP; 7.6 mg+5% sodium chloride, USP; ata pH of about 6.6 to about 7; in an appropriate amount of water forinjection (e.g., about 1 ml or about 2 ml). In one embodiment, a liquidcomposition comprising an ADIr-PEG comprises histidine—HCl, and incertain embodiments, the composition buffer is from about 0.0035 MHistidine-HCl to about 0.35 M Histidine-HCl. In one particularembodiment, the composition is formulated in a buffer comprising 0.035 MHistidine-HCl at pH 6.8 with 0.13 M sodium chloride. In anotherembodiment, the composition is formulated in a buffer comprising 0.02 Msodium phosphate buffer at pH 6.8 with 0.13 M sodium chloride.

In one embodiment, a composition comprising ADIr or ADIr-PEG has a pH ofabout 5 to about 9, about 6 to about 8, or about 6.5 to about 7.5. Insome embodiments, the composition comprising ADIr has a pH of about6.8±1.0.

In one embodiment, free PEG in a composition comprising ADIr-PEG isbetween 1-10%, and in a further embodiment, is less than 7%, less than6%, less than 5%, less than 4%, less than 3%, less than 2% or less than1% of the total PEG. In certain embodiments, the unmodified ADIr in acomposition comprising ADIr-PEG is less than about 1%, 0.9%, 0.8%, 0.7%,0.6%, 0.5%, 0.4%, 0.3%, 0.2% or less than 0.1%. Generally, compositionscomprising ADIr-PEG have total impurities less than or equal to about4%, 3%, 2%, 1.5%, 1% or 0.5%. In one embodiment, the endotoxin limitmeets the requirements stated in USP, i.e., <50 EU/mL.

In one embodiment, the free sulfhydryl in a composition comprising ADIror ADIr-PEG is greater than about 90%. In some embodiments, the freesulfhydryl in a composition comprising ADIr or ADIr-PEG is about 91%,about 92%, about 93%, about 94% or about 95%, about 96% about 97%, about98% about 99% or more.

In one embodiment, the ADIr or ADIr-PEG in a composition has a Km offrom about 0.1 μM or 0.5 μM to about 15 μM, and in a further embodiment,is from about 1 μM to about 12 μM, about 1 μM to about 10 μM, about 1.5μM to about 9 μM, about 1.5 μM to about 8 μM or about 1.5 μM to about 7μM. In certain embodiments, the ADIr or ADIr-PEG in a composition has aKm of about 1.0 μM to about 10 μM or about 1.5 μM to about 6.5 μM. Insome embodiments, the ADIr or ADIr-PEG in a composition has a Km ofabout, at least about, or less than about 0.1 μM, about 0.5 μM, about1.0 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM,about 6.5 μM, or about 7 μM, or about 8 μM, or about 9 μM, or about 10μM.

In one embodiment, the ADIr or ADIr-PEG in a composition has a Kcat offrom about 0.5 sec⁻¹ to about 80 sec⁻¹, or about 0.5 sec⁻¹ to about 70sec⁻¹, or about 0.5 sec⁻¹ to about 60 sec⁻¹, or about 0.5 sec⁻¹ to about50 sec⁻¹, or about 0.5 sec⁻¹ to about 40 sec⁻¹, or about 0.5 sec⁻¹ toabout 30 sec⁻¹, or about 0.5 sec⁻¹ to about 20 sec⁻¹, or about 0.5 sec⁻¹to about 15 sec⁻¹, and in a further embodiment, is from about 0.5 sec⁻¹to about 80 sec⁻¹, or about 1 sec⁻¹ to about 80 sec⁻¹, or about 5 sec⁻¹to about 80 sec⁻¹, or about 10 sec⁻¹ to about 80 sec⁻¹, or about 20sec⁻¹ to about 80 sec⁻¹, or about 30 sec⁻¹ to about 80 sec⁻¹, or about40 sec⁻¹ to about 80 sec⁻¹, or about 50 sec⁻¹ to about 80 sec⁻¹, orabout 60 sec⁻¹ to about 80 sec⁻¹, or about 70 sec⁻¹ to about 80 sec⁻¹,or about 1 sec⁻¹ to about 12 sec⁻¹, about 1 sec⁻¹ to about 10 sec⁻¹,about 1.5 sec⁻¹ to about 9 sec⁻¹, about 2 sec⁻¹ to about 8 sec⁻¹ orabout 2.5 sec⁻¹ to about 7 sec⁻¹. In certain embodiments, the ADIr orADIr-PEG in a composition has a Kcat of about 2.5 sec⁻¹ to about 7.5sec⁻¹. In some embodiments, the ADIr or ADIr-PEG in a composition has aKcat of about or at least about 2.5 sec⁻¹, about 3 sec⁻¹, about 3.5sec⁻¹, about 4 sec⁻¹, about 4.5 sec⁻¹, about 5 sec⁻¹, about 5.5 sec⁻¹,about 6 sec⁻¹, about 6.5 sec⁻¹, about 7 sec⁻¹, about 7.5 sec⁻¹ or about8 sec⁻¹, about 10 sec⁻¹, about 15 sec⁻¹, about 20 sec⁻¹, about 25 sec⁻¹,about 30 sec⁻¹, about 35 sec⁻¹, about 40 sec⁻¹, about 45 sec⁻¹, about 50sec⁻¹, about 55 sec⁻¹, about 60 sec⁻¹, about 65 sec⁻¹, about 70 sec⁻¹,about 75 sec⁻¹, about 80 sec⁻¹, about 85 sec⁻¹, about 90 sec⁻¹, about 95sec⁻¹, or about 100 sec⁻¹.

In one embodiment, the ADIr or ADIr-PEG in a composition has aconductivity (also referred to in the art as specific conductance) ofabout 5 mS/cm to about 20 mS/cm, and in further embodiments, from about5 mS/cm to about 15 mS/cm, about 7 mS/cm to about 15 mS/cm, about 9mS/cm to about 15 mS/cm or about 10 mS/cm to about 15 mS/cm. In someembodiments, the ADIr or ADIr-PEG in a composition has a conductivity ofabout 9 mS/cm, about 10 mS/cm, about 11 mS/cm, about 12 mS/cm or about13 mS/cm, about 14 mS/cm or about 15 mS/cm. In certain embodiments, theADIr or ADIr-PEG in a composition has a conductivity of about 13mS/cm±1.0 mS/cm.

In one embodiment, the ADIr or ADIr-PEG in a composition has anosmolality of about 50 mOsm/kg to about 500 mOsm/kg, about 100 mOsm/kgto about 400 mOsm/kg, about 150 mOsm/kg to about 350 mOsm/kg, about 200mOsm/kg to about 350 mOsm/kg or about 250 mOsm/kg to about 350 mOsm/kg.In certain embodiments, the ADIr or ADIr-PEG in a composition has anosmolality of about 300±30 mOsm/kg.

In one embodiment, the protein concentration is about 11.0±1.0 mg/mL. Incertain embodiments, the protein concentration is between about 8 andabout 15 mg/mL. In another embodiment, the protein concentration isabout 8, 9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 15 mg/mL.

In one embodiment, the specific enzyme activity is between about 5.0 and90 IU/mg or between about 5 and 55 IU/mg, where 1 IU is defined as theamount of enzyme that converts one μmol of arginine into one μmol ofcitrulline and 1 μmol of ammonia in one minute at 37° C. and the potencyis 100±20 IU/mL. In another embodiment, the specific enzyme activity isabout 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26,26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 35, 40, 45, 50, 55, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100±2.0 IU/mg. In one particularembodiment, the specific enzyme activity is 9±2.0 IU/mg.

Compositions comprising ADIr-PEG of the present disclosure may also beadministered simultaneously with, prior to, or after administration ofone or more other therapeutic agents, including ADI-PEG 20. Suchcombination therapy may include administration of a singlepharmaceutical dosage formulation which contains a compound of theinvention and one or more additional active agents, as well asadministration of compositions comprising ADIr-PEG (e.g., ADIr-PEG 20)of the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, ADIr-PEG as describedherein and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Similarly, ADIr-PEG as described herein and the otheractive agent can be administered to the patient together in a singleparenteral dosage composition such as in a saline solution or otherphysiologically acceptable solution, or each agent administered inseparate parenteral dosage formulations, by the same or different routes(e.g., one by injection, one by oral). Where separate dosageformulations are used, the compositions comprising ADIr-PEG and one ormore additional active agents can be administered at essentially thesame time, i.e., concurrently, or at separately staggered times, i.e.,sequentially and in any order; combination therapy is understood toinclude all these regimens.

Thus, in certain embodiments, also contemplated is the administration ofthe ADIr compositions of this disclosure in combination with one or moreother therapeutic agents. Such therapeutic agents may be accepted in theart as a standard treatment for a particular disease state as describedherein, such as a particular cancer or GVHD. Exemplary therapeuticagents contemplated include cytokines, growth factors, steroids, NSAIDs,DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics,autophagy inhibitors, or other active and ancillary agents.

In certain embodiments, the ADIr compositions disclosed herein may beadministered in conjunction with any number of chemotherapeutic agents.Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin. Further chemotherapeutic agentsinclude sorafenib and other protein kinase inhibitors such as afatinib,axitinib, bevacizumab, cetuximab, crizotinib, dasatinib, erlotinib,fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib,nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib,trastuzumab, vandetanib, vemurafenib, and sunitinib; sirolimus(rapamycin), everolimus and other mTOR inhibitors. Pharmaceuticallyacceptable salts, acids or derivatives of any of the above are alsocontemplated for use herein.

In certain embodiments, the ADIr compositions disclosed herein may beadministered in conjunction with any number of autophagy inhibitors. Insome preferred embodiments, the autophagy inhibitor is selected from thegroup consisting of: chloroquine, 3-methyladenine, hydroxychloroquine(Plaquenil™), bafilomycin A1, 5-amino-4-imidazole carboxamide riboside(AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibitprotein phosphatases of type 2A or type 1, analogues of cAMP, and drugswhich elevate cAMP levels, adenosine, N6-mercaptopurine riboside,wortmannin, and vinblastine. In addition, antisense or siRNA thatinhibits expression of proteins essential for autophagy, such as forexample ATGS, may also be used.

In one embodiment, the combination of ADIr-PEG with one or moretherapeutic agents acts complementary, additively, or synergistically.In this regard, complementary or synergizing agents are describedherein, which include a therapeutic agent (e.g., chemotherapeutic agent,autophagy inhibitor, mTOR inhibitor, or any other therapeutic agent usedfor the treatment of cancer, GVHD, or inflammatory bowel disease asdescribed herein) that is capable of acting complementary orsynergistically with ADIr-PEG as provided herein, where suchcomplementarity or synergy manifests as a detectable effect that isgreater (i.e., in a statistically significant manner relative to anappropriate control condition) in magnitude than the effect that can bedetected when the chemotherapeutic agent is present but the ADIr-PEGcomposition is absent, and/or when the ADIr-PEG is present but thechemotherapeutic agent is absent. Methods for measuring synergy andcomplementarity are known in the art (see e.g., Cancer Res Jan. 15, 201070; 440).

The compositions comprising ADIr, and optionally other therapeuticagents, as described herein may be used in therapeutic methods fortreating cancer and methods for preventing metastasis of a cancer. Thus,the present invention provides for methods for treating, amelioratingthe symptoms of, or inhibiting the progression of or prevention of avariety of different cancers. In another embodiment, the presentdisclosure provides methods for treating, ameliorating the symptoms of,or inhibiting the progression of GVHD. In particular the presentdisclosure provides methods for treating, ameliorating the symptoms of,or inhibiting the progression of a cancer or GVHD in a patientcomprising administering to the patient a therapeutically effectiveamount of ADIr composition as described herein, optionally, followingtreatment with ADI-PEG 20, particularly where a patient developsanti-ADI-PEG 20 antibodies, thereby treating, ameliorating the symptomsof, or inhibiting the progression of the cancer or GVHD. Thus, the ADIrcompositions described herein may be administered to an individualafflicted with inflammatory bowel disease (e.g., Crohn's disease;ulcerative colitis), GVHD or a cancer, including, but not limited tohepatocellular carcinoma, leukemia (e.g. acute myeloid leukemia andrelapsed acute myeloid leukemia), melanoma including metastaticmelanoma, sarcomas (including, but not limited to, metastatic sarcomas,uterine leiomyosarcoma), pancreatic cancer, prostate cancer (such as,but not limited to, hormone refractory prostate cancer), mesothelioma,lymphatic leukemia, chronic myelogenous leukemia, lymphoma, small celllung cancer, breast cancer, ovarian cancer, colorectal cancer, gastriccancer (including, but not limited to, gastric adenocarcinoma), glioma,glioblastoma multi-form, retinoblastoma, neuroblastoma, non-small celllung cancer (NSCLC), kidney cancer (including but not limited to renalcell carcinoma), bladder cancer, uterine cancer, esophageal cancer,brain cancer, head and neck cancers (including, but not limited to,squamous cell carcinoma of the head and neck; cancer of the tongue),cervical cancer, testicular cancer, gallbladder, cholangiocarcinoma, andstomach cancer.

In another embodiment, the present disclosure provides a method oftreating, ameliorating the symptoms of, or inhibiting the progression ofa cancer in a patient comprising administering to the patient acomposition comprising ADIr, and optionally one or more othertherapeutic agents, as described herein, wherein the cancer is deficientin ASS, ASL, or both. In this regard, ASS or ASL deficiency may be areduction in expression as measured by mRNA expression or proteinexpression, or may be a reduction in protein activity, and generallycomprises a statistically significant reduction in expression oractivity as determined by the skilled person. Reduced ASS or ASLexpression or activity may be a reduction in expression or activity ofabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, or more, as compared to expression or activity in an appropriatecontrol sample known to be cancer free. In certain embodiments, ASS orASL expression or activity is reduced by at least twofold as compared toexpression or activity in a non-cancer control sample.

In certain embodiments, the reduced expression or activity of ASS or ASLresults from methylation of the ASS or ASL promoter or inhibition of theASS or ASL promoter. In another embodiment the reduction in expressionor activity of ASS or ASL results from a DNA mutation (e.g., one or morepoint mutations, small deletions, insertions, and the like) or achromosomal abnormality resulting in deletion of the gene. In oneembodiment, the cancer is ASS or ASL negative, meaning no expression oractivity is observed.

Reduction in ASS or ASL expression or activity may be measured using anymethods known in the art, such as but not limited to, quantitative PCR,immunohistochemistry, enzyme activity assays (e.g., assay to measureconversion of citrulline into argininosuccinate or conversion ofargininosuccinate into arginine and fumarate), and the like.

Thus, the present invention provides methods for treating, amelioratingthe symptoms of, or inhibiting the progression of a cancer in a patientcomprising administering to the patient a composition comprising ADIr asdescribed herein, wherein the cancer exhibits reduced expression oractivity of ASS or ASL, or both, wherein the cancer includes, but is notlimited to hepatocellular carcinoma, leukemia (e.g. acute myeloidleukemia and relapsed acute myeloid leukemia), melanoma includingmetastatic melanoma, sarcomas (including, but not limited to, metastaticsarcomas, uterine leiomyosarcoma), pancreatic cancer, prostate cancer(such as, but not limited to, hormone refractory prostate cancer),mesothelioma, lymphatic leukemia, chronic myelogenous leukemia,lymphoma, small cell lung cancer, breast cancer, ovarian cancer,colorectal cancer, gastric cancer (including, but not limited to,gastric adenocarcinoma), glioma, glioblastoma multi-form,retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC),kidney cancer (including but not limited to renal cell carcinoma),bladder cancer, uterine cancer, esophageal cancer, brain cancer, headand neck cancers (including, but not limited to, squamous cell carcinomaof the head and neck; cancer of the tongue), cervical cancer, testicularcancer, gallbladder, cholangiocarcinoma, and stomach cancer.

Various studies in the literature have shown that ASS is deficient inthe following tumors: acute myelogenous leukemia (AML), bladder, breast,colorectal, gastric, glioblastoma, HCC, lymphoma, melanoma,mesothelioma, non-small cell lung, ovarian, pancreatic, prostate, renal,sarcoma, and small cell lung. Accordingly, treatment of theseASS-deficient cancers is specifically contemplated herein, with ADIr-PEGalone or in combination with other treatments, including treatment firstwith ADI-PEG 20.

The present invention further provides methods for treating,ameliorating the symptoms of, or inhibiting the progression of cancer ina patient comprising administering to the patient a compositioncomprising ADIr as described herein (e.g; ADIr-PEG and in particularADIr-PEG 20), in combination with an autophagy inhibitor. In oneembodiment, the present invention provides methods for treating cancerin a patient comprising administering to the patient a therapeuticallyeffective amount of a composition comprising ADIr as described herein incombination with autophagy inhibitor wherein the cancer is pancreaticcancer or small cell lung cancer.

In certain embodiments, the present invention provides methods oftreatment where administration of the compositions comprising ADIrdescribed herein depletes arginine in the plasma for at least one month,2 months, 3 months, 4 months, 5 months, 6 months or longer. In anotherembodiment, the present invention provides methods of treatment whereadministration of the compositions comprising ADIr described hereindepletes arginine in the plasma for at least one month, 2 months, 3months, 4 months, 5 months, 6 months or longer after terminatingtreatment with ADI-PEG 20 following detection of anti-ADI-PEG 20antibodies.

EXAMPLES Example 1 Screening and Selection of ADI Enzymes that have LowCross-Reactivity with Patient Anti-ADI-PEG 20 Antibodies

This example describes the screening and selection of ADI enzymes thathave low cross-reactivity with patient anti-ADI-PEG 20 antibodies.

From a large number of ADI enzymes, Table 1 lists 24 ADI enzymesselected for their sequence percent identity relative to M. hominis ADI.From the literature, M. hominis, M. arginini, and M. arthritidis ADIamino acid sequences are closely related and these enzymes have goodcatalytic properties. More recently, additional ADI enzymes have beendiscovered that have sequences closely related to these three. Moredistantly related Mycoplasma ADI enzymes have been identified, althoughless is known about them. Even more distantly related ADI enzymes frombacterial and other sources exist.

TABLE 1 Selected ADI Sequences with Varying Degrees of Similarity to M.hominis ADI SEQUENCE PERCENT ACCESSION SEQ ORGANISM IDENTITY NUMBER IDNO: Mycoplasma hominis 100.0% gi | 728876 1 Mycoplasma phocicerebrale82.1% gi | 154184333 2 Mycoplasma arginini 82.1% gi | 728875 3Mycoplasma arthritidis 80.4% gi | 238692486 4 Mycoplasma orale 77.8% gi| 2170494 5 Mycoplasma gateae 76.8% gi | 148361415 6 Mycoplasma phocidae75.3% gi | 154184335 7 Mycoplasma columbinum 58.2% gi | 343491689 8Mycoplasma iowae 55.2% gi | 350546321 9 Mycoplasma crocodyli 52.3% gi |291600396 10 Mycoplasma fermentans 52.0% gi | 238809916 11 Mycoplasmapenetrans 51.7% gi | 26554060 12 Mycoplasma gallisepticum 51.5% gi |31544533 13 Mycoplasma alligatoris 50.8% gi | 292552899 14 Mycoplasmapneumoniae 50.7% gi | 440453687 15 Mycoplasma mobile 47.3% gi | 4745838716 Streptococcus pyogenes 37.7% gi | 15675444 17 Enterococcus faecalis37.1% gi | 60389809 18 Mycoplasma capricolum 36.6% gi | 83319656 19Halothermothrix orenii 34.8% gi | 254803235 20 Staphylococcus aureus33.8% gi | 123549453 21 Pseudomonas plecoglossicida 28.7% gi | 15418375522 Pseudomonas putida 27.5% gi | 431801013 23 Pseudomonas aeruginosa27.0% gi | 452183609 24 Mycobacterium bovis 26.8% gi | 378770764 25

Several of the protein sequences available in the public databases maynot have been full-length ADI sequences. In those cases, the publiclyavailable sequences were extended where needed to make full-length ADIbased on known ADI sequences. In certain cases, the ADI proteins weremodified elsewhere (e.g., C251S substitution). These synthesized ADIsequences are provided in SEQ ID NOs:26-32 and correspond to theextended and/or modified ADI sequences of Mycoplasma arginini (C251S),Mycoplasma arthritidis (C251S), Mycoplasma phocicerebrale, Mycoplasmagateae, Mycoplasma phocidae, H. orenii, and Mycobacterium bovis.

A number of ADI enzymes from a variety of organisms were characterizedto determine which enzymes would be expected to remove and maintain lowconcentrations of arginine in patient blood, even in the presence ofanti-ADI-PEG 20 antibodies. Table 2 (below) lists a selected subset ofADI enzymes from Table 1 that were studied. As detailed below, the datafrom these studies show that ADI from a number of species that areclosely related to M. hominis, based on sequence identity, havesufficiently good enzyme catalytic properties and reducedcross-reactivity with anti-ADI-PEG 20 antibodies.

ADI Preparation. Recombinant ADI enzymes were cloned, expressed, andpurified for testing according to standard protocols, as described, forexample, in Gallego et al., PLOS One, 7(10):e47886, 2012; Monstadt andHolldorf, Biochem. J. 273:739-745, 1990; Joo Noh et al., Molecules andCells. 13:137-143, 2002; and Sugimura et al., Infection and Immunity.58:2510-2515, 1990.

Human Anti-ADI-PEG20 Antibody Purification. Anti-ADI-PEG20 antibody waspurified from plasma samples of patients who had received ADI-PEG20during a clinical study. A total of 60 ml of plasma was pooled from 8different patients that had reached high titer (titer>/=4) againstADI-PEG20 as determined by an ELISA assay. A two-step purification wasused, a Protein “A” chromatography (GE Healthcare) followed by an ADIaffinity chromatography. ˜20 mg of purified antibody was obtained andstored at −80° C. in aliquots until needed.

ADI Enzyme Assays. Arginine deiminase (ADI) catalyzes the conversion ofL-arginine to L-citrulline and ammonia. The amount of L-citrulline canbe detected by a colorimetric endpoint assay (see, for example, Knippand Vasak, Analytical Biochem. 286:257-264, 2000) and compared to astandard curve of known amounts of L-citrulline in order to calculatethe specific activity of ADI expressed as IU/mg of protein. One IU ofenzyme activity is defined as the amount of enzyme that produces 1 μmolof citrulline per minute at the pH and temperature being tested.Standard assay conditions were performed at 37° C. in PhysiologicalHEPES Buffer (PHB) 50 mM HEPES, 160 mM NaCl pH 7.4 (Lang and Zander,Clin Chem Lab Med. 37:563-571, 1999) plus 0.1% BSA. All samples andstandards were run in duplicate or triplicate where conditionspermitted.

Km and Kcat values were determined by using a variation of the activityassay described above. As with the activity assay, all reactions wererun at 37° C. in PHB plus 0.1% BSA. Enzyme concentration, reaction time,and substrate concentration range were adjusted for each of the ADI orADIr constructs to account for their differences in activity. Ingeneral, 2 nM enzyme, 5 minute reaction time, and a 0-160 μM argininewas used as starting conditions. When optimizing the conditions,particular attention was paid towards the amount of substrate consumedas a percentage of total substrate added to the reaction. The lowerlimit of detection is 1 μM of citrulline with the lower limit ofquantitation being 2 μM. A citrulline standard curve was run on everyplate and used to quantify the citrulline produced by the enzymaticreaction.

Activity assays were also performed to assess enzymatic activity in thepresence of anti-ADI-PEG20 (antibody neutralization profiles). Theseassays were performed as described above and in the presence of 800 nM,400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 0 nM ofanti-ADI-PEG20 antibodies.

Calculations. The citrulline concentration (μM) produced in eachreaction well was calculated and averaged using the citrulline standardcurve. The velocity of each reaction was then calculated in μM/min/50 nMADI. Specific activity (IU/mg or μmols product/min/mg ADI) wascalculated by multiplying this value by the “IU” factor (IU factor wascalculated from the molecular weight of the ADI and the reactionvolume). The results are summarized in Table 2 below.

TABLE 2 Selected ADI Sequences and Properties NUMBER OF SEQUENCESPECIFIC SURFACE REDUCED AB PERCENT ACTIVITY RESIDUE CROSS- ORGANISMIDENTITY (IU/MG)*** CHANGES* REACTIVITY**** Mycoplasma hominis 100.0 + 0— Mycoplasma phocicerebrale 82.1 + 33 Y Mycoplasma arginini 82.1 + 50 YMycoplasma arthritidis 80.4 + 53 Y Mycoplasma gateae 76.8 + 48 YMycoplasma phocidae 75.3 + 51 Y Mycoplasma columbinum 58.2 + 93 YMycoplasma iowae 55.2 − 107 ND Mycoplasma crocodyli 52.3 − 107 NDMycoplasma gallisepticum 51.5 − 108 ND Mycoplasma alligatoris 50.8 + 111Y Mycoplasma mobile 47.3 ND 111 ND Halothermothrix orenii 34.8 − 122 NDMycobacterium bovis 26.8 − 132 ND *Surface residues were identified fromthe crystal structure of M. hominis ADI and surface residues for ADIfrom other organisms was determined by sequence homology. ** ND: NotDetermined. ***“+” indicates specific enzymatic activity of ≥8 IU/mgunder physiological conditions and in the absence of anti-ADI-PEG20antibodies. ****“Y” indicates reduced anti-ADI-PEG20 cross-reactivityrelative to M. hominis ADI, as measured by specific enzymatic activityin the presence of anti-ADI-PEG20 antibodies.

These data show that ADI enzymes that are highly homologous to M.hominis ADI (about 50-100 percent identity) maintained excellentcatalytic activity. They also showed reduced affinity toward patientanti-ADI-PEG 20 antibodies, as measured by enzyme activity in thepresence of anti-ADI-PEG 20 antibodies, for example, relative to that ofMycoplasma hominis. Accordingly, these ADI enzymes may have therapeuticutility for use in therapy for the treatment of cancer, either alone orfollowing ADI-PEG 20 treatment, to extend and/or increase theeffectiveness of arginine depletion therapy.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, application and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1.-28. (canceled)
 29. An isolated arginine deiminase, or a fragmentthereof having ADI activity, wherein the isolated arginine deiminase hasreduced cross-reactivity with patient anti-ADI-PEG 20 antibodies. 30.The isolated arginine deiminase of claim 29 wherein the isolatedarginine deiminase is not from M. hominis.
 31. The isolated argininedeiminase of claim 30 wherein the isolated arginine deiminase is from anorganism listed in Table
 1. 32. The isolated arginine deiminase of claim29 wherein the isolated arginine deiminase has one or more propertiescomparable to or better than those of ADI-PEG
 20. 33. The isolatedarginine deiminase of claim 32 wherein the one or more properties isKeat, Km, pH optimum, stability, in vivo proteolytic stability, or norequirement for ions or cofactors that are not already present in blood,or any combination thereof.
 34. The isolated arginine deiminase of claim29 wherein the isolated arginine deiminase has at least 20 surfaceresidue changes as compared to M. hominis arginine deiminase.
 35. Theisolated arginine deiminase of claim 29 wherein the isolated argininedeiminase has between 20 and 135 surface residue changes as compared toM. hominis arginine deiminase.
 36. The isolated arginine deiminase ofclaim 29 wherein the isolated arginine deiminase has between 40 and 100surface residue changes as compared to M. hominis arginine deiminase.37. The isolated arginine deiminase of claim 29 wherein the isolatedarginine deiminase has between 30 and 60 surface residue changes ascompared to M. hominis arginine deiminase.
 38. The isolated argininedeiminase of claim 29 wherein the isolated arginine deiminase hasbetween 80 and 100 surface residues changes as compared to M. hominisarginine deiminase.
 39. The isolated arginine deiminase of claim 29wherein the isolated arginine deiminase has between 100 and 120 surfaceresidues changes as compared to M. hominis arginine deiminase.
 40. Theisolated arginine deiminase of claim 29 wherein the isolated argininedeiminase is from M. arginini, M. arthritidis, M. phocicerebrale, M.gateae, M. phocidae, M. columbinum, M. iowae, M. crocodyli, M.alligatoris, H. orenii, or M. bovis.
 41. The isolated arginine deiminaseof claim 29 wherein the isolated arginine deiminase comprises the aminoacid sequence set forth in any one of SEQ ID NOs:2-32.
 42. The isolatedarginine deiminase of claim 29 wherein the isolated arginine deiminasehas been modified to remove at least one pegylation site.
 43. Theisolated arginine deiminase of claim 29 wherein at least one lysineresidue has been modified by an amino acid substitution.
 44. Theisolated arginine deiminase of claim 43 wherein at least 5 lysineresidues have been modified by an amino acid substitution.
 45. Theisolated arginine deiminase of claim 43 wherein at least 10 lysineresidues have been modified by an amino acid substitution.
 46. Theisolated arginine deiminase of claim 43 wherein at least 15 lysineresidues have been modified by an amino acid substitution.
 47. Theisolated arginine deiminase of claim 43 wherein at least 20 lysineresidues have been modified by an amino acid substitution.
 48. Theisolated arginine deiminase of claim 29 wherein the arginine deiminaseis covalently bonded via a linker to a PEG molecule.
 49. The isolatedarginine deiminase of claim 48 wherein the arginine deiminase iscovalently bonded to more than one PEG molecule.
 50. The isolatedarginine deiminase of claim 48 wherein the arginine deiminase iscovalently bonded to about 1 to about 10 PEG molecules.
 51. The isolatedarginine deiminase of claim 48 wherein the arginine deiminase iscovalently bonded to about 2 to about 8 PEG molecules.
 52. The isolatedarginine deiminase of claim 48 wherein the PEG molecules are straightchain or branch chain PEG molecules.
 53. The isolated arginine deiminaseof claim 48 wherein the PEG has a total weight average molecular weightof from about 1,000 to about 40,000.
 54. The isolated arginine deiminaseof claim 48 wherein the PEG has a total weight average molecular weightof from about 10,000 to about 30,000.
 55. The isolated argininedeiminase of claim 48 wherein the linker is a succinyl group, an amidegroup, an imide group, a carbamate group, an ester group, an epoxygroup, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosinegroup, a cysteine group, a histidine group, a methylene group, or anycombinations thereof.
 56. The isolated arginine deiminase of claim 55wherein the source of the succinyl group is succinimidyl succinate. 57.A polynucleotide encoding the isolated arginine deiminase of claim 29.58. A vector comprising the polynucleotide of claim
 57. 59. An isolatedhost cell comprising the vector of claim
 58. 60.-68. (canceled)